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Determination of free N-acetylneuraminic acid in edible bird nest: A development of chemical marker for quality control



Abbreviations: NANA: N-acetylneuraminic acid, EBN: Edible bird's nest, LC-MS/MS, QQQ: Triplequadrupoles liquid chromatography tandem mass spectrometry Abstract Edible bird nest (EBN) is a common health food consumed in Asia. The exaggerated skincare functions resulted in the abnormally high market value. Fake EBN started to appear in the market for the sake of its profits. A reliable authenticate method is in urgent need. Here, the determination of a free form of sialic acid, N-acetylneuraminic acid (NANA), was developed to distinguish EBN according to their grading. High amount of free NANA was revealed in red and yellow EBN: both white and grass EBN contained free NANA at low level. Unlike total NANA content measurement, fake EBN did not show detectable amount of free NANA. Moreover, the water extract of EBN showed anti-tyrosinase activity, and which was in line to amount of free NANA. Thus, the amount of free NANA could differentiate fake EBN from the genuine one, as well as its grading. In conclusion, the quantitation of free NANA by triplequadrupoles liquid chromatography tandem mass spectrometry (LC-MS/MS QQQ) was shown to be the best method in EBN authentication. In addition, the amount of free NANA in EBN was in accord to anti-tyrosinase activity of EBN.
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The Journal of Ethnobiology and Traditional Medicine. Photon 120 (2013) 620-628
Original Research Article. ISJN: 6642-3194
The Journal of Ethnobiology and Traditional Medicine
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Determination of free N-acetylneuraminic acid in edible bird nest: A
development of chemical marker for quality control
Gallant Kl Chan
, Ken Yz Zheng
, Kevin Y Zhu
, Tina Tx Dong
, Karl Wk Tsim
Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and
Technology, Clear Water Bay Road, Hong Kong, China
Hanshan Normal University, Chaozhou, Guangdong 521041, China
Article history:
Received: 4 February, 2013
Accepted: 15 February, 2013
Available online: 26 October, 2013
NANA: N-acetylneuraminic acid, EBN: Edible bird’s nest,
LC-MS/MS, QQQ: Triplequadrupoles liquid
chromatography tandem mass spectrometry
Aerodramus fuciphagus, Edible Bird Nest, LC-MS/MS
QQQ, Authentication, Quality control, Sialic acid; Anti-
Corresponding Author:
Tsim K.Wk.*
Phone: 85223587332
Fax No: 85223581559
Edible bird nest (EBN) is a common health food
consumed in Asia. The exaggerated skincare
functions resulted in the abnormally high market
value. Fake EBN started to appear in the market for
the sake of its profits. A reliable authenticate
method is in urgent need. Here, the determination
of a free form of sialic acid, N-acetylneuraminic acid
(NANA), was developed to distinguish EBN
according to their grading. High amount of free
NANA was revealed in red and yellow EBN: both
white and grass EBN contained free NANA at low
level. Unlike total NANA content measurement, fake
EBN did not show detectable amount of free NANA.
Moreover, the water extract of EBN showed anti-
tyrosinase activity, and which was in line to amount
of free NANA. Thus, the amount of free NANA could
differentiate fake EBN from the genuine one, as well
as its grading. In conclusion, the quantitation of free
NANA by triplequadrupoles liquid chromatography
tandem mass spectrometry (LC-MS/MS QQQ) was
shown to be the best method in EBN authentication.
In addition, the amount of free NANA in EBN was in
accord to anti-tyrosinase activity of EBN.
Chan G. Kl., Zheng K. Yz., ZhuK.Y., Dong T. Tx., Tsim
K.Wk., 2013. Determination of free N-acetylneuraminic
acid in edible bird nest: A development of chemical marker
for quality control. The Journal of Ethnobiology and
Traditional Medicine. Photon 120, 620-628.
1. Introduction
1.1 Adulteration of edible bird’s nest due to the
lacking of quality control marker
Edible bird nest (EBN) has been served as a
valuable delicacy in Asia for over 1,000 years.
The official record of EBN has only been found
since 16th century from ancient Chinese
literatures. According to the traditional
application, the intake of EBN could repair
lung function, strengthen digestive system,
enhance skin repairing and improve immune
system (Zhao, 1765); however, neither clinical
trial nor basic mechanistic study has proven
those beneficial effects. Although the
functional role of EBN is still largely unknown,
it becomes a very popular health food
supplement for its exaggerated skincare
function promoted by the media. Recently,
fake EBN appeared commonly in the market
(Leung, 2004). The unscrupulous traders may
illegally sell EBN adulterants in the market,
e.g. white fungus and pig skin. On the other
hand, the market prices of EBN varied by
grading of EBN. The grading of EBN mainly
depends on the genuine color, e.g. red, yellow
and white, and the packaging forms, e.g. cup,
stripe and piece. EBN contains impurities such
as seaweeds (grass) and feathers will be
determined as lower grade. Apart from
grading, the scarcity of specific type of EBN
produced from those Southeast Asian
countries such as Vietnam, Malaysia and
Thailand also seriously affects the market
price of EBN in Hong Kong. Refer to the
previous report, the market price of EBN is
ranged from $2,000 to $10,000 USD per
kilogram (Ma & Liu, 2012). The disparity in
market price between different grades of EBN
is even more obvious now. For each kilogram
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of EBN, the local price of Grass EBN ranged
from $1,000 to $2,000 USD. For Indonesian
EBN, White EBN ranged from $1,000 to
$3,000 USD, Yellow EBN ranged from $1,500
to $3,500 USD and Red EBN ranged from
$2,000 to $4,000 USD. The price for EBN from
Thailand and Malaysia reaches $6,500 and
7,500 respectively. The high quality EBN from
Vietnam even reached $15,000 USD per
kilogram. Some of the traders may modify the
colour appearance of EBN as to achieve a
higher price. Currently, microscopic and
genetic methods were being applied in
authenticating EBN (Lin et al., 2006b; Lin et
al., 2009). However, the ability in
differentiating different grading of EBN was in
doubt. Thus, a fast and reliable method for
EBN authentication and/or classification is
urgently needed.
1.2 N-acetylneuraminic acid as a potential
quality marker for EBN
EBN comes from the nest of swiftlet species,
such as Aerodramus fuciphagus (formerly
named Collocalia fuciphagus), which is made
by their saliva secretion (Green, 1885). EBN
composes of up to 60 % of soluble protein and
about 20 % of water, which also contains fat,
carbohydrate and trace amount of minerals
including sodium, calcium and magnesium
(Norhayati et al., 2010). More importantly,
EBN consists of ~9% of sialic acid (Kathan et
al., 1969). Sialic acid is a family of more than
20 compounds derived from neuraminic acid,
and N-acetylneuraminic acid (NANA) is the
predominant form (Ham et al., 2007). Due to
the highly charged property, NANA is involved
in retaining water on cell surface and
enhances cellular fluid uptakes (Simons &
Fuller, 1985). Moreover, NANA is proposed to
be a major component for anti-influenza
function of EBN (Guo et al., 2006; Yagi et al.,
2008),as well as its effect on the proliferation
of Caco-2 cells (Rashed & Nazaimoon, 2010).
Thus, NANA could serve as a good quality
control marker.
The majority of NANA exists in conjugated
form (i.e. oligosaccharides and glycoproteins).
After the actions of neuraminidase or acid
hydrolysis, a free form of NANA will be
released, and the function of free NANA is
unclear (Sillanaukee et al., 1999).Different
EBN authentication methods have been
developed by quantifying the amount of NANA
(Yu et al., 1998; Huang et al., 2003; Wang et
al., 2006), but all of these methods are using
total amount of NANA (i.e. sum of conjugated
and free NANA) as an authentication marker.
Unfortunately, the conjugated NANA can be
found in many other food sources including
dairy products (Wang et al., 2001). Using total
NANA as a marker for EBN authentication will
be resulted in a highbackground and obtained
larger false positive error. Another drawback of
the current established EBN authentication
methods is that they are incapable of
differentiating different grades of EBN.
2. Objective of Research
Several methods in authenticating EBN such
as electron microscopy (Lin et al., 2006b) and
DNA sequence analysis (Lin et al., 2009) are
proven to be failed in differentiating different
grades of EBN. Thus, total NANA and free
NANA are now under examination as quality
marker of EBN.
2. Materials and Methods
2.1 Chemicals
NANA was purchased from Sigma-Aldrich (St.
Louis, MO), and 10 mM was used as standard
stock solution. The volume was measured
accurately from the stock, and diluted with
fresh Milli-Q water to produce a series of
solution standards (1, 2, 5, 10, 15, 20 µM).
Tyrosinase (EC from mushroom
and L-3, 4-dihydroxyphenyl-alanine (L-DOPA)
was from Sigma-Aldrich. The specific activity
of the enzyme was 1,000 U/mg.
2.2 LC-MS/MS QQQ system
The liquid chromatograph is equipped with an
Agilent 6410 Triple Quad MS/MS (Agilent,
Waldbronn, Germany), and an Eclipse XDB-
C18 column (2.1 X 100 mm; 3.5 µm particle
size). The injection volume was 2 µL. A 5 min
linear gradient at flow rates of 0.4 ml/min
between solvent A (Milli-Q water, 0.1% formic
acid) and solvent B (acetonitrile, 0.1% formic
acid) was used. After reaching 80% B, the
system returned to 100% A in 0.5 min. For
column equilibration, a total cycle time of 10
min was needed. Retention time of NANA was
at 0.65 – 0.69 min. The MS was operated in
negative electron spray ionization mode. A
capillary voltage of 3.5 kV and a cone voltage
of 10 V were applied. The source temperature
was 100° C, and the desolvation temperature
was 325° C. Ultra high purity nitrogen was
used for cone gas (3.0 L/min), desolvation gas
(10.0 L/min) and nebulising gas (35 psi). For
collision induced dissociation (CID), collision
energy of 5 eV was used. Negatively single
charged ions [M-H]- of NANA (m/z 307.9) were
selected as precursor ions for CID. The
precursor ion was dissociated into two major
product ions (m/z 87.0 and 170.0), and the
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product ions m/z 87.0 was the most abundant
from NANA. No internal standard was applied
in this study as the reference for free NANA
was limited, and the relevant chemical could
not be easily accessible. 7 For multiple
reaction monitoring, the transitions m/z 307.9
87.0 and m/z 307.9 170.0 were chosen
as the qualifiers, whilst the transitions m/z
307.9 87.0 was measured for quantification.
For the full scanning of total ion
chromatograms, spectra from m/z 50 to m/z
1000 were recorded.
2.3 Quantification of free NANA
EBN samples were grounded into powder
(approximately 1 3 mm) and mixed
thoroughly. 10 mg of each was weighed, and
free sialic acid was extracted by 1 ml of fresh
Milli-Q water under sonication for 10 min.
Followed by centrifugation at full speed for 5
min, the supernatants were filtered, and the
filtrates were collected for LC-MS/MS QQQ
analysis. A 5-point calibration curve with
concentrations of 1, 2, 5, 10 and 20 µM NANA
was made. All calibrators were prepared in
fresh Milli-Q water. Triplicate results were
taken for each sample.Genuine Indonesia
white EBN was used for method validation.
Linearity of free NANA was tested using the
calibration curve of NANA. All calibrators were
prepared in fresh Milli-Q water. The correlation
coefficient, better than 0.9992, was obtained.
2.4 Total NANA assay
The total NANA content was determined by
sialic acid quantitation kit - SIALICQ (Sigma).
Ten mg of powder EBN were weighed
accurately in screw cap microtubes, 0.5 ml of
1 M sulphric acid in methanol was added to
the each tube. The tubes were then heated to
90° C for 90 min in a heating block, and then
which was cool down in room temperature for
10 min. Barium carbonate (0.5 g) was added
to neutralize the solution. The tube was then
sonicated for 5 min and spin down at 1,500 Xg
for 5 min. Supernatants were transferred into
clean tubes and evaporated to dryness at 37
oC. The tubes were then reconstituted by 600
µL of pH 7.5 Tris-HCl, and 1 µL of NANA
aldolase was added. The tubes were
incubated at 37° C for 16 hours. The solution
was diluted 5 times by Tris-HCl buffer at pH
7.5, and a diluted buffer was measured as
blank. Then, 20 β-NADH was added to the
each solution. The solution was mixed well,
and the absorbance reading at 340 nm was
recorded from the initial reading to the final
reading. The NANA content was calculated by
the equation provided in the kit.
2.5 Tyrosinase activity assay
The activity of mushroom tyrosinase was
monitored by dopachrome formation at 492
nm through the oxidation of L-DOPA. The
reaction medium (200 µl) contained 0.5 mM L-
DOPA in 50 mM sodium phosphate buffer (pH
6.8), 5 mM vitamin C was used as a control
inhibitor. The final concentration of mushroom
tyrosinase was 0.2 mg/ml. In this method, 0.1
ml of different concentration of effectors,
including EBN extracts, adulterants of EBN
and NANA, was added to the reaction. The
reaction mixtures were loaded on a 96-well
plate, and the formation of dopachrome was
measured in optical density at 492 nm after 20
min of incubation under dark. The reaction
was carried out under room temperature.
Absorption was recorded using micro-plate
2.6 Statistical tests
Statistical tests were done by using student t
test and one-way ANOVA provided in
GraphPad Prism 5.0. Statistically significant
changes were classed as [*] where p<0.05, [**]
where p<0.01 and [***] where p<0.001.
3. Results
3.1 Authentication methods of EBN
Figure 1: Photos of different grades of EBN
Grass, white, yellow and red EBN are the
major grades of EBN commonly found in the
market (Fig. 1): they are greatly different in
their price, i.e. red > yellow > white > grass.
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Under normal circumstance, EBN could be
easily differentiated by their physical
appearance. However, different ways of
mimicking red EBN by white EBN had been
developed by illegal traders for better profit.
The amount of total NANA was reported to
serve as a quality control marker of EBN
(Wang et al., 2006). Here, we aimed to
determine the amounts of total NANA in EBN.
Fig. 2 shows the amount of total NANA in
different grades of EBN, and they were in an
order of red > yellow > white > grass EBN.
However, the commonly used dopants, or the
fake EBN, showed significant high amount of
total NANA. Thus, total NANA could not be
served as an authenticating marker here, in
particular to identify genuine EBN.
Figure 2: Amount oftotal NANA in EBN and its
The amount of total NANA content was
extracted by water from 27 batches of genuine
EBN (i.e. grass EBN (n=3), white EBN (n=9),
yellow EBN (n=6) and red EBN (n=9) and 21
batches of known fake EBN (n=6) and five
adulterants of EBN (n=3) by SIALIC-Q kit. The
total NANA in mg/kg of dry material was
presented as Mean + SEM (n ± 3).
3.2 Free NANA for EBN authentication
In order to search possible chemical markers
for EBN, the extracts deriving from EBN and
its common dopants were subject to LC-
MS/MS QQQ analysis. The representing total
ion chromatograms of different grading of EBN
and EBN adulterants/dopants (agar, gelatin
and pig skin) were achieved (Fig. 3A). The
retention time of all characteristic peaks from
those different samples were compared. The
mass spectrums from each peak were also
analyzed as a reference for identity. From the
result, all EBN chromatograms showed
distinguishable peak 1 at the retention time of
0.523 – 0.538 min, but peak 1 was also found
in pig skin at the same retention time. From
the mass spectrum information, peak 1 could
be an alanine-rich compound. On the other
hand, a well separated peak 2 at 0.655 min
was noticed in EBN chromatograms. The
mass spectrum suggested a compound having
an ion of m/z 307.9: this was identified as
NANA (Fig. 3A). The NANA peak (peak 2) in
the grass EBN was merged into peak 1 due to
its low level. A characteristic peak 3 was only
observed in red EBN, and the mass
spectrometry result revealed that as an
unknown compound with a m/z 191.0. In
conclusion, an identified free form of NANA
could be considered as a chemical marker of
The characteristics of NANA in the MS/MS
were revealed (Table 1). Two product ions
were produced here, 87 and 170 m/z: the ion
at 87 m/z was used for quantitation
(Supplementary Fig. 1). In MS/MS analysis,
each calibration curve was obtained from
different concentrations of marker chemical.
The correlation coefficient (r
) of the calibration
curve was higher than 0.999. The LOD and
LOQ were determined at 3.188 µg/kg and
10.626 µg/kg, respectively. The precision,
repeatability and recovery were determined as
described previously (Zhu et al., 2010), and
the results were satisfactory (Table 1). The
results showed that the LC-MS/MS QQQ
method was precise, accurate and sensitive
enough for simultaneous, quantitative
evaluation of NANA from EBN.
Figure 3: The chromatographic fingerprint of EBN
and its adulterants
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(A): Different water extracts of EBN and its
adulterants, as shown in the figure, were
detected using the MRM scan mode in
MS/MS. The ions with m/z 50 to m/z 1000
were recorded. Ions fractions acquired at
specific time were marked with different
numbers according to their compositions.
Peak 1 is unknown small peptide, peak 2 is
corresponding to NANA and peak 3 is
unknown. Representative chromatograms are
shown, n=3.
(B): The amount of free NANA in different
grades of EBN (i.e. grass EBN (n=3), white
EBN (n=9), yellow EBN (n=6) and red EBN
(n=9)) and its adulterants (n=21) were
determined. Free NANA was extracted from
10 mg of each sample by 1 ml MilliQ water
and measured by LC-MS/MS QQQ system.
The amount of free NANA in µg/kg of dry
material was presented as Mean + SEM (n
After the establishment and validation of our
EBN authentication method, the quality of EBN
available in Hong Kong market was examined.
The extraction of free NANA from EBN was
optimized at sonication for 10 min in water
(Supplementary Fig. 2).
Table 1: Mass spectra properties and results of validation tests on the assay of free NANA in EBN
Mass spectra properties Validation tests results
Formula C
Calibration curve
Calculated mass [M] 309.1 Correlation coefficient (r
) 0.9992
Precursor ion [M-H]
307.9 Linear range (ng) 0.001 – 0.050
Fragmentor energy
135 V
Collison energy
5 eV Intra-day precision (n=6)
RSD (%) 4.24
Product ion
87.0, 170.0 Inter-day precision (n=6)
RSD (%) 4.57
Retention time (min)
0.672 Repeatability (n=5) RSD (%) 0.66
LOD f (
g/kg) 3.188 Reproducibility (n=5) RSD (%) 3.35
LOQ g (µg/kg) 10.626 Recovery
(n=5) Mean (%) 87.33
RSD (%) 7.34
The detected chemicals had the greatest responses under the negative mode: the [M-H]- was used as the
precursor ion.
The fragmentor energy was optimized to have the greatest ionize efficiency.
The collision energy was optimized to have the greatest product ion intensity, which was the key factor in the
MRM mode.
Two product ions were used for the MRM analysis. The upper one was used for quantitative analysis and the
lower one was for qualitative analysis, which could guarantee the precision of analytes.
The retention time was determined by 5 different individual analyses (n = 5).
LOD refers to the limits of detection.
LOQ refers to the limits of quantification.
These calibration curves were constructed by plotting the peak area versus the concentration of each analyte.
Each calibration curve was derived from eight data points, n = 3.
The intra-day analysis refers to the sample examined for six replicates within one day.
The inter-day analysis refers to the sample examined in duplicates over three consecutive days.
Recovery (%)=100×(amount found−original amount)/amount spiked. The data was presented as average of five
independent determinations, and the SD was < 8% of the Mean, which was not shown for clarity.
The free NANA was determined in EBN and
commonly reported EBN dopants, including
agar, white fungi, pig skin, tora and gelatin.
The free NANA was not detected in EBN
dopants/adulterants (Fig. 3B). In contrast,
NANA was revealed in all EBN having an
order of red > yellow > white > grass EBN. The
median of the free NANA in different EBN
groups were 702.51 µg/kg, 351.44 µg/kg,
170.46 µg/kg and 54.31 µg/kg respectively.
In the market, EBN was clustered according to
their form of packaging (Fig. 4A). EBN sold in
a form of their original “bird’s nest” shaped
were regarded as “cup”; EBN comprised of
long stripes broke down horizontally from the
nest were regarded as stripe”; and EBN sold
in the form of powder-like or small pieces
within 5-mm diameter were regarded as
“piece”. No significant differences in the
content of free NANA between different forms
of packaging was observed among white EBN
(Fig. 4B). In contrary, the differences in
amount of free NANA between varies
packaging forms of red EBN was obvious. Cup
EBN showed the highest content of free
NANA, followed by the stripe EBN, and the
piece EBN contained the lowest content of
free NANA.
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Figure 4: Free NANA in different packaging forms
of EBN
(A): Photos of different packaging forms of EBN.
Scale bar, 10 mm
(B): The amount of free NANA in different
packaging forms of EBN was determined. 18 EBN
purchased from Hong Kong market were
categorized into three forms of packaging (i.e. piece
EBN, stripe EBN and cup EBN) from white (n=9)
and red EBN (n=9). Free NANA was extracted from
10 mg of each sample by 1 ml MilliQ water and
measured by LC-MS/MS QQQ system. The amount
of free NANA in µg/kg of dry material was
presented as Mean + SEM (n 3). Statistical
significant differences were indicated. *** P < 0.001
versus reference group.
3.3 EBN inhibits tyrosinase activity
The inhibitory effect of EBN on tyrosinase
could serve as an indicator for skin whitening
function. Both white and red EBN inhibited the
formation of dopachrome and reduced the
content by at least 50% (Fig. 5). As compared
to the control, a slight reduction of
dopachrome content was found with the
extract of grass EBN; however, the difference
was not statistical significant. None of the
adulterants of EBN could inhibit tyrosinase
activities. Oppositely, the extracts from agar or
pig skin enhanced the oxidation of L-DOPA. In
parallel, free NANA was able to inhibit the
tyrosinase activity, even though the
concentration used was rather high as
compared to that contained within EBN (Fig.
5). The application of vitamin C served as a
Figure 5: Anti-tyrosinase activity of EBN
The dopachrome formed by the oxidation of
0.5 mM of L-DOPA incubated for 20 min with
0.2 mg/ml mushroom tyrosinase was served
as control. Vitamin C (5 mM) was served as an
inhibitor control. The dopachrome
concentration, after treatment of EBN or
others, was determined, all at 10 mg / ml water
extract. Free NANA was at 20 mM. The
contents were presented as Mean + SEM (n =
3). Unpaired one-tailed student t test was
performed on the data set by Graph Pad 5.0.
Statistical significant differences were
indicated. *** P < 0.001 versus control group.
4. Discussions
The authentication methods of EBN can be
divided into four major directions including
physical examination, DNA authentication,
proteomics analysis and glycan and monose
determination. The physical examination on
EBN was mainly based on different ways of
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microscopy (Lin et al., 2006b). The
advantages of this method are that the nature
of EBN can be maintained or with minimized
interruption, and also no extraction procedure
is required. However, this EBN authentication
is qualitative and not able to distinguish the
grading of EBN.
DNA fragment has been used for EBN
authentication (Lin et al., 2009; Aowphol et al.,
2008). The DNA sequence in EBN is unique,
thus these authenticating properties will not be
cheated easily. However, the DNA
authentication could not reflect the actual
nutrient composition in EBN. Moreover, the
resolution power of using DNA authentication
is not enough for differentiating EBN grading
(Lin et al., 2006b).
Proteomics analysis has been developed to
study EBN including 1-D (Lin et al., 2006a)
and 2-D polyacrylamide gel electrophoresis
(Goh et al., 2001). On the other hand,
epithelial growth factor-like activity (Kong et al.
1987) and mitogenic activities (Ng et al., 1986;
Roh et al., 2011; Hou et al., 2010; Fadhilah et
al., 2011) of EBN were reported. In addition,
the effects of EBN on bone and skin have
been reported (Matsukawa et al., 2011). All of
those reports suggested that a hormone or
growth factor–like substance in protein nature
should be found in EBN. Recently, an antibody
raised against an EBN glycoprotein was
employed for authentication (Zhang et al.,
2012). The database of proteomics of EBN still
under developed, and only a few proteins have
been identified in EBN so far (Goh et al., 2000;
Ou et al., 2001). Moreover, the protein
identification could not classify the grading of
Many different types of specific glycans have
been reported to be presented on EBN surface
(Yagi et al., 2008; Oda et al., 1998; Nakagawa
et al., 2007). Glycan was proposed to
responsible for the biological functions of EBN,
e.g. anti-influenza effect (Guo et al., 2006).
Indeed, five sugar markers, including
mannose, galactose, N-acetylgalactosamine,
N-acetylglucosamine and total NANA, were
introduced in EBN. The detection of these
sugar markers however was unable to
differentiate different types of EBN (Yu et al.,
Here, we introduce a new EBN authentication
method by revealing the level of free NANA in
EBN. Actually, scientists discovered free form
of NANA in EBN since 1960s (Howe et al.,
1961) andthe application of free NANA
quantitation was widely used by diagnostic
study (Ham et al., 2007).However, it is the first
report in combining the two distinct areas of
research and accurately quantifying the free
form of NANA by advance technology for the
authentication of traditional Chinese medicine
or nutraceuticals. The method could be done
within 30 min, and which showed highly
repeatable and reproducible. More important,
this method distinguished genuine EBN as
well as to classify the grading of different EBN.
The form of packaging of EBN is another
parameter that varies the market price
seriously. The intact EBN having “nest
shaped” shows the highest price: this is also
named as cup EBN. According to history in
China, only cup EBN could be used by the
king of Southeast Asian countries as gifts to
Chinese Empires, thus which was named as
officer EBN. As supported by our studies, the
free NANA content in cup EBN is the highest
among different forms of packaging, which
matches with the traditional classification. This
difference could only be revealed here in red
EBN: white EBN, according to packaging
forms, was not significant. The processing of
EBN may be one of the possible reasons in
reducing the amount of free NANA, which
involves the removal of feather and other
foreign matters, air-drying and baking. These
processing steps for strip and pieces of EBN
require a long period of time of water soaking
and molding. In order to preserve white EBN
color after longer time of storage, white EBN
was subjected to additional hot air drying or
chemical spraying (Law et al., 2011). Free
NANA is highly water soluble and degrades
rapidly under acidic environment, and thus
free NANA could be decreased after prolong
processing, as suggested previouslyYu et al.,
1998). In contrast, red cup EBN has lesser
extent of processing.
The skincare functions of EBN have been
widely accepted by the public; however, there
is no scientific supporting evidence until now.
In the past, the unproven skincare functions of
EBN was relied mainly on the high protein
content and the finding of the epidermal
growth factor-like activity (Kong et al., 1987).
Here, we provided a direct report to show the
potential skin whitening function of EBN, and
the adulterants of EBN cannot replace EBN.
Hence, EBN with higher quality (e.g. red EBN)
may have better whitening effect than EBN
with low quality (e.g. grass EBN). Interesting,
the whitening function of EBN was in parallel
to the amount of free NANA. The action
Ph ton
mechanism of NANA on the skincare functions
should need further investigation.
Honestly speaking, all authentication methods
have advantages but also drawbacks on their
own. Like other drugs with only single quality
assurance marker, free NANA could be
externally added on fake EBN by those
dishonest traders.
Thus, a combination of multi-disciplinal
methods including genomic analysis,
microscopic identification and analytical
analysis should be applied together to
establish a quality control of EBN.
Definitely, due to the correlation of the
bioactivity, this result strengthens the role of
NANA as a quality control marker of EBN.
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... Edible bird's nest (EBN), or cubilose, is a health food supplement originated from salivary secretion by specific swiftlets, mainly from Aerodramus fuciphagus and Aerodramus maximus (Kang et al., 1991), which has been proven to have nutritious and therapeutic values, such as anti-influenza viruses, antioxidant, skin lightening, bone strength improvement, anti-inflammatory, and epidermal growth enhancement (Kong et al., 1987;Kong et al., 1989;Guo et al., 2006;Aswir and Wan Nazaimoon, 2011;Matsukawa et al., 2011;Yew et al., 2014; Besides, most of the publications still retained in elucidating chemical composition as the quality control parameters: since no official method has been established for quality surveillance of EBN (Deng et al., 2006;Wang et al., 2006;Wu et al., 2010;Chan et al., 2013a). ...
... n = 6. 2007), total proteins (Hu and Lai, 1999;Liu et al., 2012;Zhang et al., 2012), N-acetylneuraminic acid (Chan et al., 2013a), sialic acid (Wang et al., 2006), DNA (Wu et al., 2010), and sodium nitrate (Chan et al., 2013b) of EBN have been established; but no official method could be found for quality control of EBN by specific protein analysis, regardless of its abundance. Here, we utilized SGF digestion and HPLC to generate the peptide fingerprint, which could authenticate EBN on a qualitative and quantitative basis robustly and therefore provide a comprehensive picture in quality control of EBN. ...
Full-text available
OBJECTIVES Proteins are the major component and play a key role in nutritious and therapeutic functions of edible bird’s nest (EBN); however, limited studies have been conducted on the protein due to difficulties in extraction, isolation as well as identification. This study aimed to provide comprehensive information for the quality evaluation of EBN peptides, which would be a valuable reference for further study on EBN proteins. METHODS Here, we developed a quality control method using high performance liquid chromatography (HPLC) peptide fingerprints deriving from EBN being digested with simulated gastric fluid. The characteristic peptide peaks were collected and identified by LC-MS/MS. RESULTS The characteristic peptide peaks, corresponding to the protein fragments of acidic mammalian chitinase-like, lysyl oxidase, and Mucin-5AC-like, were identified and quantified. Interestingly, the principal component analysis indicated that the fingerprints were able to discriminate colour of EBN (white/red) and production sites (cave/house) of White EBN on the same weight basis. As proposed by the model developed in this study, Muc-5AC-like and AMCase-like proteins were the markers with the highest discriminative power. CONCLUSIONS The overall findings suggest that HPLC peptide fingerprints were able to clearly demonstrate peptide profile differences between genuine and adulterated EBN samples; and classify EBN samples by its color and production site. In addition, the protein identification results suggested that Muc-5AC-like protein was the major protein in EBN.
... The approach was enhanced by specific chemometric analysis, which resulted in convincing and reliable data [10] in anticipation of very high market response and fake EBNs starting to appear in the market. So, the problem of authentication is also very necessary [11]. ...
Full-text available
Indonesia is the first exporter of edible bird's nest commodities to China. Therefore, the Agricultural Quarantine Agency is one of the agencies responsible for ensuring the fulfilment of the requirements requested by China. One of these conditions is the upper limit of nitrite content in edible bird's nest commodities. This paper contains the results of research aimed at developing a spectral-based sensor to detect the upper limit of nitrite content and quantitatively measure nitrite content in edible bird's nest commodities. This spectral sensor is applied to the inspection process for edible bird's nest commodities which must meet export requirements to China. The objective of this research is to find the spectrums capable of significantly measuring nitrite levels around the required maximum value of 30 PPM. The experimental methodology started by testing 18 spectrum channels to study their effect on nitrite content in edible bird's nest commodities. The spectrum channels used are in the range from 410 nm to 940 nm with an accuracy of ±12%. The edible bird's nest commodity that was tested, was pre-treated by pre-conditioning it by making it into a powder so that the level of homogeneity of the nitrite measurement in the edible bird's nest commodity increased. The results showed that three response frequencies were the most dominant and had a major influence on the results of the measurement of nitrite content in edible bird's nest commodities around the required maximum value of 30 PPM. The three spectrum channels have wavelengths values of w1=410 nm, w2=435 nm, and w7=560 nm. The three wavelengths mathematically have a linear relationship to the nitrite value measured with an average error value of about 1.88%.
... Normally the sialic acid exist in conjugated form such as oligosaccharides and glycoprotien. However, after hydrolysis of EBN, the neuraminic acid or acetylmuramic acid will be released (Chan et al., 2013). ...
Full-text available
Type 2 diabetes mellitus is characterized by both resistance to the action of insulin and defects in insulin secretion. Bird’s nest, which is derived from the saliva of swiftlets are well known to possess multiple health benefits dating back to Imperial China. However, it’s effect on diabetes mellitus and influence on the actions of insulin action remains to be investigated. In the present study, the effect of standardized aqueous extract of hydrolyzed edible bird nest (HBN) on metabolic characteristics and insulin signaling pathway in pancreas, liver and skeletal muscle of db/db, a type 2 diabetic mice model was investigated. Male db/db diabetic and its euglycemic control, C57BL/6J mice were administered HBN (75 and 150 mg/kg) or glibenclamide (1 mg/kg) orally for 28 days. Metabolic parameters were evaluated by measuring fasting blood glucose, serum insulin and oral glucose tolerance test (OGTT). Insulin signaling and activation of inflammatory pathways in liver, adipose, pancreas and muscle tissue were evaluated by Western blotting and immunohistochemistry. Pro-inflammatory cytokines were measured in the serum at the end of the treatment. The results showed that db/db mice treated with HBN significantly reversed the elevated fasting blood glucose, serum insulin, serum pro-inflammatory cytokines levels and the impaired OGTT without affecting the body weight of the mice in all groups. Furthermore, HBN treatment significantly ameliorated pathological changes and increased the protein expression of insulin, and glucose transporters in the pancreatic islets (GLUT-2), liver and skeletal muscle (GLUT-4). Likewise, the Western blots analysis denotes improved insulin signaling and antioxidant enzyme, decreased reactive oxygen species producing enzymes and inflammatory molecules in the liver and adipose tissues of HBN treated diabetic mice. These results suggest that HBN improves β-cell function and insulin signaling by attenuation of oxidative stress mediated chronic inflammation in the type 2 diabetic mice.
... Edible bird's nest (EBN) is produced by the secretion of swiftlets Aerodramus fuciphagus (white-nest swiftlets) and Aerodramus maximus (blacknest swiftlets) during breeding season. EBN or 'Yan Wo' in Chinese is a traditional cuisine and delicacy among the Chinese society, mainly due to its rich nutrient content such as sialic acid, carbohydrate, amino acid and epidermal growth factor-like protein (Chan et al., 2013;Chua et al., 2013;Lee et al., 2015). Traditionally, EBN is harvested in caves, a natural habitat of the swiftlets. ...
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There is an urgent need to develop a rapid and robust approach to ensure traceability of the Edible Bird’s Nest (EBN) products. The main composition of EBN is protein and it plays an important role in EBN. This study shows that there are significant differences in the protein profile of EBN collected from different states in Malaysia. The SDS-PAGE method is used to analyse the protein profile for 65 samples collected from 13 different states of Malaysia. SDS-PAGE gel image showed that all EBN samples analyzed have been well separated between protein bands. Data shows that protein band at 212 kD can only be detected in state P, while 135 kD can only be found in state M. EBN from other states have their unique combination of fingerprints that made them different from one to another. This research demonstrated the possibility of using SDS-PAGE pattern for identification and differentiation of the geographical origins of EBN produced in Malaysia. These findings are the first of its kind in EBN analysis.
... ). The result agreed with the previous studies that N-acetylneuraminic acid (NANA) is the predominant form of sialic acid in EBN[33][34][35]. The ...
Full-text available
Background and Aim: Edible bird's nest (EBN) is the nutrient-rich salivary bioproduct produced by swiftlets in Southeast Asia. Currently, researchers are exploring the therapeutic effects of EBN, such as cell growth promotion, antioxidant content, antiviral effects, bone strengthening, eyes care, and neuroprotection bioactivities. The therapeutic effects of EBN have been studied through different extraction methods but the metabolites profile of the EBN in each extract has not yet been elucidated. This study aimed to profile the water-soluble metabolites of EBN prepared in different extraction methods. Subsequently, an extraction method will be selected as an ideal extraction method for untargeted metabolite profiling on the water-soluble metabolites in EBN. Materials and Methods: In this study, water-soluble metabolites of EBN extracted by the four extraction methods were subjected to metabolite profiling through liquid chromatography-mass spectrometry (LC-MS). The extraction methods were acid extraction(ABN), pancreatic extraction (EzBN), eHMG extraction, and spray drying of HMG extraction (pHMG). The metabolite profiles, such as the number of metabolites and their identities in each extraction method, were evaluated through LC-MS analysis. Results: The identity of metabolites present in the four extraction methods is inconsistent. Based on LC-MS analysis, only one and six metabolites were extracted differently through EzBN and ABN, respectively, in the first pre-screening. Through the second LC-MS screening on pHMG and eHMG extraction methods, eHMG was selected as an ideal extraction method due to the highest numbers of water-soluble metabolites with an amount of 193 was detected. Besides, eHMG extraction method was able to extract sialic acid and a high percentage of secondary metabolites. Conclusion: This study suggests that eHMG is the ideal extraction method for extracting higher number of water-soluble metabolites from EBN and could be further developed as an extraction method for industry application. In addition, this study also has identified the types of primary and secondary metabolites present in EBN.
... Due to the abundance of N-linked glycans present on EBN [7], the major form of NANA belonged to conjugated form, and thus in general only a trace amount of free NANA was detected on EBN. The amount of free form of NANA has been proposed to be a quality control marker for authentication and categorization of EBN in different grading [8]. At that period of time, microscopic [9] and genetic [10] methods were two commonly used strategies for EBN authentication. ...
... In the past, various detection methods were established in distinguishing fake and authentic EBN and still have been used until today. For example, EBN samples were examined using gas chromatographic method (GC) to detect five monoses (Dmannitose, D-galactose, N-acetyl-Dgalactosamine, N-acetyl-D-glucosamine, and N-acetyl neuraminate) which can be regarded as EBN's fingerprint (Chan et al., 2013;Marcone, 2005;Yu-Qin et al., 2000). Anatomical traits were observed, and protein bands used as a measurement to determine adulterant in EBN's sample (Hu et al., 1999;Lin et al., 2009;Wu et al., 2010). ...
Full-text available
Edible bird's nest (EBN) originates from South East Asia and has become the most popular food delicacy among Chinese people. The price for raw and clean EBN are around RM 4,000 and RM 8,000 per kg, respectively. Due to the high market value of EBN and low maintenance cost, people ventures and invest in bird's nest industry. The nesting house resembles the cave is built to attract swiftlets to roost and nest in it in order to meet the world's growing demand. Out of the blue, in 2011, the EBN's price falls half from its original price from RM 4,000 to RM 2,000. Nitrate found in raw or unclean EBN is one of the reason price plunges and the public also worried about counterfeit EBN that made from unclear substances. Thus, this review paper aims to highlight the nutritional properties in EBN that give medicinal benefits to consumers and the adulterant in EBN to increase the property contents with the dangerous mechanism by some irresponsible producers to obtain better profit which can threatening public's health. In relation, these matters may influence consumer's perception on EBN contents that could harm this industry. For that reason, scientific studies determined ways of handling EBN from scratch and distinguished the adulterant in EBN to reduce consumer anxiety was also discussed. Strict standard operation procedure enforces by the Malaysian Ministry of Health to farmers and producers are required, so that Malaysia EBN would appeal to the China market. Latter it can yield a good result and the price of EBN will slowly increase.
... Due to the abundance of N-linked glycans present on EBN [7], the major form of NANA belonged to conjugated form, and thus in general only a trace amount of free NANA was detected on EBN. The amount of free form of NANA has been proposed to be a quality control marker for authentication and categorization of EBN in different grading [8]. At that period of time, microscopic [9] and genetic [10] methods were two commonly used strategies for EBN authentication. ...
... EBN has been consumed by the populations in China and Southeast Asian countries for its skincare function, especially for skin lightening, for over 800 years. The biological functions of EBN were not only well recorded in ancient Chinese literatures, but also reported in a list of scientific publications in previous years [5] [6] [7] [8]. EBN was not the only natural health food which recorded in the ancient Chinese literature for skin- care functions. ...
Full-text available
Skin lightening and tanning are two major areas dominating the market of skincare and cosmetic products. Though the demands are originated from two different communities, the two areas share a same goal-skin colour tuning. The known safe compounds with skin colour tuning activities are limited. In contrary, Chinese medicinal herb provides a pool of natural bioactive compounds, which have been used in Asian countries for long time and have been tested for its toxicity. Here, we demonstrate a high throughput screening platform for potential compounds usable for skin colour tuning. From 147 natural compounds, 26 of them showed potential in skin tanning functions by using the high throughput melanogenesis platform based on the melanogenesis assay on B16 melanocytes. Five of them promoted melanogenesis by over 50%. Moreover, apart from 1, 8-dihydroxyanthraquinone, the other four compounds showed enhancement effect on tyrosinase activity. From the result, the compounds increased the Vmax of tyrosinase without changing the Km in a dose-dependent manner. Thus, there should be no irreversible structural change of the enzyme. Definitely, this report contributes to the development of personalization in skincare and cosmetic products.
... Nutritional and physicochemical analyses are the most frequently used method for determination of EBN production and geographical origins (Chua, Chan, et al., 2014;Huda et al., 2008;Saengkrajang et al., 2013). Sialic acid remains nowadays as the unique indicator, which allows grading of different EBN, while antioxidant parameters allow explanation of the functional effects of EBN (Chan, Zheng, Zhu, Dong, & Tsim, 2013;Yang, Cheung, Li, & Cheung, 2014). ...
Full-text available
Malaysian edible bird's nests (EBN) are from the swiflet species, Aerodromus fuciphagus. The objective of this study was to determine and compare the nutrient composition of EBN obtained from different parts of Peninsular Malaysia, collected at three different harvesting seasons, to four commercial brands. A total of 18 raw, unprocessed EBN samples from the North, South and East Coast zones of Peninsular Malaysia and duplicate samples of 4 commercial brands (processed) of EBN samples were analysed. The protein and mineral contents of unprocessed EBN samples between zones and harvesting seasons were comparable. Mean (± SEM) protein content of unprocessed EBN was 61.5 ± 0.6 g/100g and the top four minerals detected were calcium, sodium, magnesium and potassium with mean (± SEM) concentration of 553.1 ± 19.5 mg/100g, 187.9 ± 10.4 mg/100g, 92.9 ± 2.0 mg/100g and 6.3 ± 0.4 mg/100g respectively. Sialic acid content ranged between 0.7 to 1.5%, and remained comparable between samples from different zones and harvesting seasons. The commercial brands were found to contain higher amounts of calcium, sodium, magnesium, potassium and phosphorus compared to unprocessed EBN, warranting further investigation and verification with more samples. Since the nutrient contents of EBN may be affected by seasonal variations and even breeding sites, it is recommended that a more comprehensive study be conducted involving more samples and breeding sites as such data are important to ensure sustainability of the EBN industry in this country.
Objective: To research on the ConA induced lymphocytes transformation of rats and its promoting effect stimulated by Edible birds&apos; nest (short for EBN). Methods: Chose three parameters: cell concentration, ConA concentration and culture period to establish MTT ...
The edible bird's nest is a nest made from the saliva of swiftlets (Aves: Apodidae). It is highly acclaimed as a catholicon and is a comprehensive health food. Increasing desire for the edible bird's nest makes the investigation of the nests urgent. There is a broad and growing interest in knowing more about the components and the nutritional and medicinal values of the edible bird's nest. This review discusses the research on the edible bird's nest currently, especially the research on its bioactivities. The content of water-soluble proteins, carbohydrates, inorganic salts, and various kinds of elements shows the important nutritional value of the edible bird's nest. The reported bioactivities and nutritional value of the edible bird's nest include the potential for mitogenic response, epidermal growth factor (EGF)-like activity, anti-influenza virus, hemagglutination-inhibitory activity, lectin-binding activity, improvement of bone strength and dermal thickness, and hormone content etc. In the future, more scientific work should be done to fully elucidate the biological and medicinal functions of the edible bird's nest. The relationships of components and functions of the nest should be studied more. Bioactive components need to be isolated and purified to make full use of the edible bird's nest.
Edible bird’s nest (EBN) is a functional food constructed with swiftlets’ salivary glue. Counterfeit EBN products have been found in the market due to limited supply and high price of genuine EBN. In this article, a method for genetic identification of EBN was developed. The technique is based on sequence of cytochrome b gene in mitochondrial DNA. The sample sequences together with the sequences of swiftlets in GenBank were used to construct phylogenetic trees for genetic identification of samples. This method was applied to 11 EBN samples, one instant EBN soup product from Indonesia, and Huaiji EBN, a counterfeit EBN in some regions of China. Results showed that all the EBN samples and the instant EBN soup were from Aerodramus fuciphagus while the Huaiji EBN sample was from Apus nipalensis. This was consistent with identification based on morphology of the samples. Therefore, this method is a promising tool to identify the species of bird producing a given sample of EBN, and thus could be used to authenticate—that is, distinguish authentic from counterfeit—EBN.
The N-linked asialo carbohydrate, released by digestion treatment of the glycoprotein of the edible bird's nest of Collocalia fuciphaga, was identified as l-asparagine-linked fucose-containing triantennary oligosaccharide, composed of Galβ1→4GlcNAcβ1→4(Galβ1→4GlcNAcβ1→2)Manα1→3(Galβ1→4GlcNAcβ1→2Manα1→6)Manβ1→4GlcNAcβ1→4(Fucα1→6)GlcNAcβ1, on the basis of chemical and spectroscopic methods, in addition to GLC and HPLC analyses on achiral columns. Keywords: Collocalia fuciphaga; edible bird's nest; nest-cementing substance; pronase digestion; desialylation; N-linked fucose-containing triantennary oligosaccharide